| Baculovirus expression system(BEVS)is a eukaryotic expression system,which has many advantages such as large capacity of exogenous genes,good biological safety and complete post translation modification system,etc.The expression of secretory protein generally requires the guidance of signal peptide.And choosing the appropriate signal sequence is an important means to regulate the expression and secretion of secretory protein.In this paper,we choose a gp64 signal peptide commonly used in baculovirus expression system,and a kind of artificial designed signal peptide HMM38 reported in the literature which are used to guide the secretion and expression of eGFP.The influence of different signal peptides on protein secretion expression is explored in baculovirus expression system.Compared with the natural signal peptide gp64,the fluorescence of HMM38 signaling peptide guided protein was weak,and concentrated near the cell membrane.The insertion of signal sequence significantly reduced the total protein expression,but the secretion efficiency of protein appeared to be improved,by detecting fluorescence intensity of eGFP.Western blot results showed that synonymous mutations in HMM38 signal peptide coding sequence also affected the production of secreted proteins.The previous studies have shown that the energy of the signal peptide encoding sequence is lower than that of the non-signal peptide coding sequence.Therefore,on the basis of the artificial HMM38 signal peptide,the near codon sequence(MWWR)is replaced with the Kozak sequence MSK which is more suitable for the high expression gene of baculovirus.At the same time,the coding sequence of signal peptide was recoded and screened according to the predicted secondary structure and energy.Nine coding sequences X1-9 with different energy were selected as reference.The effects of coding sequences with different energy on protein secretion were studied by detecting the expression of protein in baculovirus expression system.The results show that the coding sequences of different energy do affect the expression of protein secretion,but the specific mechanism and law of the effect are still unclear.Bovine infectious rhinotracheitis virus(IBRV)is a kind of virus that is popular and causes great economic losses to cattle industry in China and even the world.At present,there is no effective means to prevent and control the disease caused by the virus.The results show that the envelope glycoprotein of IBRV plays an important role in the adsorption and invasion of host cells,pathogenicity,down regulation of the expression of host cell surface proteins,and virus assembly and budding.The main antigen proteins that stimulate the immune response of the body are gB and gE,which are also the main targets of host humoral immunity and cellular immunity.In the second part of this paper,four expression plasmids were constructed by using two protein labels(His and hFc)for the extracellular region of gB and gE of IBRV.The expression of gB and gE protein was carried out in baculovirus expression system.The results showed that the higher protein yield could be obtained when MOI = 0.1after infection with high five cells with different viruses MOI.After that,the same protein with different labels was paired.And the antibody was detected by colloidal gold immunochromatography.The results showed that the negative and positive serum could be distinguished,when the sample serum was diluted by 4 times.This study proves that different coding sequences of the same signal peptide can affect the secretion of protein to some extent.Recoding the signal peptide may become a technical means to regulate the expression and secretion efficiency of secreted proteins.In addition,the two proteins of IBRV,gB and gE,were successfully expressed.The gold colloidal test paper was prepared by colloidal gold immunochromatography,which laid the foundation for the establishment of a rapid and simple basic detection method for the virus. |
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